Solubilization of liver alkaline phosphatase isoenzyme during cholestasis in dogs

Objective-To determine the mechanism by which liver alkaline phosphatase (L ALP) isoenzyme is converted from a membrane-bound enzyme to the soluble enz yme during cholestasis. Sample Population-Serum and tissues from 2 dogs. Procedure-The LALP was purified by use of affinity chromatography in sample s of serum from dogs with complete bile duct obstruction. Gas chromatograph y/mass spectrometry was used to detect myo-inositol residues that would be evident when serum LALP had been membrane-attached and released by phosphol ipase activity. Exclusion chromatography, gel electrophoresis, and octyl-se pharose phase separation of the serum isolate were used to confirm cleavage of the hydrophobic membrane anchor. Western immunoblot analysis was used t o distinguish release by glycosylphosphatidylinositol phospholipase D (GPI- PLD) from that by glycosylphosphatidylinositol phospholipase C (GPI-PLC). I ntact hepatocytes were incubated with canine serum GPI-PLD to test sensitiv ity of LALP to release by GPI-PLD. Hepatocyte membrane fragments were treat ed with serum GPI-PLD and mixtures of taurocholate and taurodeoxycholate to test effects of bile acids on LALP release. Results-Amounts of myo-inositol per mole of serum LALP isolate were equal t o amounts detected with LALP isolated from hepatic tissue. Evaluation of re sults of western immunoblot analysis and electrophoretic mobility suggested release by GPI-PLD rather than by GPI-PLC. Membrane-bound LALP was resista nt to serum GPI-PLD activity in the absence of bile acids; however, incubat ion in the presence of bile acids caused release of LALP. Conclusions-Solubilization of LALP during cholestasis involves cleavage of its membrane anchor by endogenous GPI-PLD activity. Action of GPI-PLD is li kely enhanced by increased concentrations of hepatic bile acids during chol estasis.